Osteoporosis
Osteoporosis is defined as a disease characterized by low bone mass and microarchitecture deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk. The most frequent osteoporotic fractures are those of the proximal femur, distal forearm, and vertebrae. Osteoporotic vertebral fractures are associated with a significant increase in morbidity and mortality including severe and chronic back pain, functional limitation, height loss, spinal deformity, and disability.
A common occurrence in older people is compression fractures of the vertebrae that is commonly treated by vertebroplasty procedure in which cement is injected into a fractures vertebra. In his clinical procedure bone cement is injected at high pressure into the interior of a vertebral body, without the prior formation of a cavity. Vertebroplasty is an invasive procedure and has also been applied to vertebral haemangiomas and painful lesions caused by metastatic disease. An injection of the liquid cement with a cannula is made into the vertebral body. The injectate is typically a polymethyl methacrylate cement (PMMA) used more commonly to fix joint prostheses to bone. The method produces an in situ polymerization and gives immediate results on bone pain. PMMA has some disadvantages including excess heat generated during the polymerization process, and the possibility of inducing giant cell granulomas and fibrous reactions. Adjacent vertebral overload has been reported with maximal PMMA filling, possibly provoking fractures. Systems, devices and methods for placing material directly into bones are described for example in U.S. Pat. Nos. 7,153,307, 7,008,433, 6,241,734, 6,613,054 and in US publication number US 2007/0233249.
Another common treatment applicable to vertebra fractures is kyphoplasty in which a balloon is first inflated inside the vertebra that is next filled with a fixing material or an implant. This treatment reduces the risk of cement migration, yet it suffers other limitations as poor contact between the filler and the bone tissue.
Composite cements containing methacrylic polymers and HA have been proposed but all these biomaterials are not resorbable or biodegradable. Tri Calcium phosphate bone substitutes are biocompatible, bioactive, and biodegradable with osteoconductive properties.
The association of HA and beta-tri calcium phosphate (beta-TCP), in suitable proportions provides biphasic calcium phosphate (BCP) ceramics whose bioactivity depends on the HA/beta-TCP ratio. Treatment of vertebroplasty using calcium phosphate cement (CPC) alone in such patients has been reported, but complications such as recrushing of the vertebra and prolapse of the cement into the spinal canal may occur. Matsuyama et al. (2004) have showed that vertebral reconstruction with biodegradable CPC in the treatment of osteoporotic vertebral compression fracture using instrumentation (such as screws to fix adjacent vertebrae) was a safe and useful surgical treatment. Fracture fixation in osteoporosis is a critical factor since the surrounding bone is weak to begin with (Kraut 2004). The use of BCP granules in the injectable form has been evaluated in osteoporotic rats to test their potent restorative properties on bone mass and bone microarchitecture (Blouin et al. 2006). It was concluded that biomaterial trials must be conducted with long-term implantation periods, in aged osteoporotic animals.
Hollinger et al. (2007) have studied whether recombinant human platelet-derived growth factor-BB (rhPDGF-BB) delivered in an injectable beta-tricalcium phosphate/collagen matrix would enhance tibial fracture healing in geriatric osteoporotic rats.
Ilvesaro et al. (1998) described inhibition of bone resorption in vitro by a peptide containing the cadherin cell adhesion recognition sequence HAV (His-Ala-Val), and suggested that the tight attachment of osteoclasts to the bone surface in the sealing zone area may be mediated by cadherin-like molecules.
Existing agents such as estrogen, bisphosphonates, fluoride, or calcitonin can prevent bone loss and induce a 3-5% increase of bone mass by refilling the remodeling space, but net bone formation is not significantly stimulated. The retention of bone by inhibition of bone turnover may not be sufficient protection against fracture risk for patients who already have significant bone loss. It is suggested that anabolic agents that increase bone strength by stimulating bone formation preferentially may provide better protection against fracture in patients with established osteoporosis.
Known metabolites and hormones affecting osteoporosis (review by Lopes and Pereira 2007) are for example, parathyroid hormone (PTH) and its 1-34 fragment (described for example in U.S. Pat. No. 6,977,077), administered preferably by subcutaneous injection, daily in >5 microgram/Kg/day; Matrix Gla proteins (MGP) which inhibit mesenchymal differentiation into osteogenic cell lines by blocking the action of BMP (bone morphogenic proteins) inducing osteopenia; Osteopontin (OPN) a matrix protein that binds to osteoclasts through specific integrin and functions as an important inhibitor of calcification; Osteoprotegrin (OPG), a soluble cytokine of the tumor necrosis factor (TNF) receptor family produced by many cells and its absence was shown to be involved in osteoporosis and calcification of the vascular wall of the aorta and renal arteries. Other agents, such as bisphosphonates operate by preventing the resorption of bone. U.S. Pat. No. 5,280,040 discloses compounds described as useful in the treatment of osteoporosis by preventing bone resorption.
Bisphosphonates, formerly called diphosphonates are compounds characterized by two C—P bonds. There are a number of known pharmacologically active bisphosphonates including alendronate, clodronate, etidronate, ibandronate, icadronate, pamidronate, risedronate, tiludronate and zoledronate. The main effect of these pharmacologically active bisphosphonates is to inhibit resorption both in vitro and in vivo. These effects are related to the marked affinity of these compounds for solid-phase calcium phosphate, on the surface of bone. There is a general consensus that the bisphosphonates act by inhibiting the activity of osteoclasts. Osteoclasts are inhibited when they come into contact with bisphosphonates-containing bone. This supports the hypothesis that bisphosphonates are deposited onto bone because of their strong affinity for the mineral, and that the osteoclasts are then inhibited when they start to engulf bisphosphonates-containing bone.
The bisphosphonates investigated up to now appear to be absorbed, stored, and excreted unaltered in the body. Thus, bisphosphonates seem to be non-biodegradable, both in animals and in solution. The intestinal absorption lies between 1% and 10%. Between 20% and 50% of the absorbed bisphosphonate is localized to the bone, the remainder being rapidly excreted in the urine. The half-life of circulating bisphosphonates is short, in the rat only of the order of minutes and in human about 2 hours. Although the nitrogen-containing bisphosphonates such as alendronate and pamidronate, have been shown to be effective in preventing the bone loss, these drugs also appear capable of causing injury to the upper gastrointestinal tract in addition ulcerations and, especially, osteonecrosis of the jaws and their have been several case reports of severe oesophagitis in patients treated with alendronate. Alendronate has also been shown to cause erosions and ulcers in the human stomach and to interfere with the healing of pre-existing lesions when given to healthy volunteers at doses that are prescribed for the treatment of osteoporosis and Pagets disease of bone.
Tissue engineering includes the provision of cells or of a natural or synthetic scaffold that serves as an architectural support onto which cells may attach, proliferate, and synthesize new tissue to replace tissue losses due to disease, trauma or age. The trend in tissue engineering in general is to utilize biomaterials to promote healing or tissue regeneration. In orthopedics and dentistry the clinical focus transforms from traditional metal and other inorganic implants, plates, screws and cements to biologically based products for mineralized tissue regeneration. Natural polymers are of major interest in tissue engineering since they tend to be biocompatible and biodegradable and may have the potential to enhance cell adhesion and proliferation. Additionally, such material substrates can be prepared in various forms and shapes, including strips, sheets, sponges and beads for implantation.
Bone is a unique type of tissue that comprises both organic and inorganic phases, that undergoes modeling and remodeling wherein old bone is lost (resorption) and new bone is formed (formation/replacement). Bone formation may be enhanced either by recruiting osteoblasts, the bone forming cells, or by inhibiting recruitment or activity of osteoclasts, the bone resorbing cells. Osteoblasts and osteoclasts work together in a coordinated fashion to form and remodel bone tissue. The activities of these cells are regulated by a large number of cytokines and growth factors, many of which have now been identified and cloned.
There is a plethora of conditions which are characterized by the need to enhance bone formation or to inhibit bone resorption. Perhaps the most obvious is the case of bone fractures, where it would be desirable to stimulate bone growth and to hasten and complete bone repair. Agents that enhance bone formation would also be useful in facial reconstruction procedures. Other bone deficit conditions include bone segmental defects, periodontal disease, metastatic bone disease, osteolytic bone disease, osteopenia, spinal fusion and conditions where connective tissue repair would be beneficial, such as healing or regeneration of cartilage defects or injury. Also of great significance is the chronic condition of osteoporosis, including age-related osteoporosis and osteoporosis associated with post-menopausal hormone status. Other conditions characterized by the need for bone growth include primary and secondary hyperparathyroidism, disuse osteoporosis, diabetes-related osteoporosis, and glucocorticoid-related osteoporosis.
Many materials have been utilized for bone repair. Synthetic materials are being developed in order to replace autologous harvesting problems and the health risks attendant with allogeneic material. Inorganic materials such as calcium phosphate and hydroxyapatite have been utilized as bone and dental fillers (reviewed in LeGeros, 2002) but lacking many of the extra cellular like functionalities, none can be considered entirely satisfactory in meeting the criteria required for successful tissue engineering.
Biomineralization of Bone
Biomineralization refers to the deposition of inorganic solids in biological systems. The natural mineralization of bone is considered to occur by deposition of hydroxyapatite (HA, having the chemical formula Ca10(PO4)6(OH)2), or its precursor forms in an organic extracellular matrix composed of collagen and other proteins, many of which are rich in acidic residues (Hunter, 1996; Teraub, 1989). The major role of collagen is to render the bone improved mechanical properties through an hierarchical composition of the organic fibers and aligned HA minerals (Lowenstam et al., 1989, Mann, S., 2001). Non-collagenous proteins (i.e. bone sialoprotein, osteopontin, osteocalcin, osteonectin and others, Young et al, 1992), isolated from bone extracellular matrices that are rich in acidic amino acids, have been proposed to be involved in the nucleation, and growth of carbonated apatite. Among these, sialoprotein, a glycosylated and sulphated phosphoprotein, found almost exclusively in mineralized connective tissues, is the most widely accepted protein linked to apatite nucleation (Ganss et al., 1999). Sialoprotein exhibits regions rich in both glutamic- and aspartic-acid residues (Oldberg et al, 1988) as well as the cell binding arginine-glycine-aspartate (RGD) motifs. Despite numerous studies aiming at unraveling the principles of apatite biomineralzation, detailed mechanisms that account for the role of acid rich proteins in this process, are yet to be elucidated.
Among the main properties of organic interfaces that may be contributing to nucleation of biominerals are electrostatic accumulation and structural correspondence. Electrostatic accumulation is considered to be the initial step in biomineralization. It is believed that one of the most essential properties of bone acid-rich proteins and possibly also collagen is their ability to control nucleation by charged amino acid residues on their surfaces. The primary residues are acidic and phosphorylated amino acids, which at biological pH, may expose charged functional groups, i.e. negatively charged carboxylate groups of glutamic acid and aspartic acid as well as negatively charged phosphates. (Addadi, 1985; Mann, 1988).
Peptide Matrices
Recent developments in the study of peptide self-assembly matrices have advanced the understanding of the relationship between amino acid composition, molecular assembly forms and interaction of these materials with cells. Certain peptides and proteins have been shown to promote osteogenic cell adhesion. A 15-mer peptide fragment of collagen 1 α1has been designed to include cell binding domain for mesenchymal progenitor cells. This fragment is commercially available as Pepgen P-15® in combination with anorganic bovine derived bone mineral as particles or cement for bone grafting in patients with periodontal osseous defects (Valentin and Weber, 2004). Gilbert, et al. (2000) teach a fusion peptide of two extracellular matrix proteins, statherin and osteopontin that binds HA and mediates cell adhesion. The chimeric peptide was shown to have utility in tissue engineering and vaccine applications. Goldberg, et al. (2001) teach synthetic poly-L-glutamic acid and poly-L-aspartic acid peptides and their ability to bind HA. He, et al. (2003) report that the acidic protein dentin matrix protein 1 (DMP 1) assembles into acidic clusters that are claimed to nucleate the formation of HA in vitro.
International patent application WO 2005/003292 relates to a composition useful for making homogenously mineralized self-assembled peptide amphiphile nanofibers and nanofiber gels which may be prepared with appropriate phosphate and calcium solutions to yield mineral templated matrices.
U.S. Pat. Nos. 5,670,483; 5,955,343; 6,548,630 and 6,800,481 relate to amphiphilic peptides having alternating hydrophobic and hydrophilic residues, and their resultant macroscopic membranes. Specifically, two peptides having the amino acid sequences (AEAEAKAK)2 and (ARARADAD)2 were shown to self assemble into macroscopic membranes useful for in vitro culturing of cells and biomaterial applications. The former sequence was originally found in a region of alternating hydrophobic and hydrophilic residues in a yeast protein called zuotin.
US Patent Publication No. US 2005/0181973 discloses a self-assembling peptide comprising two domains, the first comprising complementary alternating hydrophobic and hydrophilic amino acids that are overall neutrally charged with equal number of positively and negatively charged amino acids, and self-assemble into a macroscopic structure, including hydroegls, when present in unmodified form; and a second domain comprises a biologically active peptide motif or a target site for an interaction with a biomolecule. That application further teaches that replacement of the positively charged residues, lysine (K) and arginine (R), by negatively charged residues, such as aspartate (D) and glutamate (E), prevents peptide self-assembly into macroscopic structures and only β-sheet and not macroscopic structures are formed in the presence of salt. The VE20 peptide, a 20-mer peptide comprising alternating valine (V) and glutamate (E) amino acids, was disclosed as not able to self-assemble to form macroscopic structures.
US Patent Publication No. US 2006/0025524 discloses a method for making a hydrogel from a solution of peptides, mainly peptides containing Val-Lys repeats or peptides with at least one positively-charged residue per 6 amino acids, which undergo change in conformation from random coil to β-hairpin secondary structures, that promote hydrogel formation. The hydrogel is formed by alteration peptide concentration or one or more environmental signals or stimuli (e.g., change in pH, ionic strength, specific ion concentration, and/or temperature of the solution).
US Patent Publication No. 2004/0120922 discloses a method for promoting bone formation by administering of amine polymers.
The “RGD” (Arg-Gly-Asp) tri-peptide sequence, which occurs in fibronectin and has been shown to promote cell adhesion and growth, has been disclosed in inter alia, U.S. Pat. Nos. 4,988,621; 4,792,525 and 5,695,997. U.S. Pat. No. 6,291,428 teaches peptides comprising the RGD amino acid sequence for promoting in situ bone mineralization.
The inventor of the present invention reported amphiphilic peptides that form β-strand monolayers when spread at air-water interfaces (Rapaport, 2000; Rapaport, 2002, WO 2007/148334). Peptides of seven to 17 amino acid residues were found to form crystalline arrays with coherence lengths of about 100 to about 1000 Å. A 30-residue peptide, which incorporates proline residues to induce reverse turns, was designed to form an ordered triple stranded β-sheet monolayer at the air water interface.
Specific methods for prevention and treatment of osteoporosis and pre-osteoporotic conditions by local administration of compositions comprising these peptides were neither taught nor suggested in those publications.
There is an unmet medical need for improved compositions and methods for prevention of progression and treatment of osteoporosis and pre-osteoporotic conditions.